Reproduction depends on production of LH and FSH from the pituitary and CG from the placenta. Expression of the genes encoding these gonadotropins occurs in a strict temporal and spatial sequence. Any insult that perturbs their expression has dire consequences on reproductive fitness. Based on our previous work, we postulate that three levels of combinatorial code are used to direct appropriate temporal and spatial expression of gonadotropin genes. The first involves formation of a cell-specific composite regulatory element from a larger menu of many regulatory elements. The second level occurs through selection of one of many transcription factors that can bind a given element. Finally, the third code utilizes specific co-adapters that link the required DNA-binding proteins to the core transcription complex.
The aims below differ from the previous funding period because we turn from cis-acting elements that comprise the first level of code to the second and third tiers that include both DNA-binding proteins and proteins that bind to them.
Aims 1 and 2, we will use the glycoprotein hormone alpha subunit gene (alphaGSU) as a model for understanding how a single gene can be targeted for expression to two cell types that do not share a common lineage and are spatially distinct. Emphasis will be placed on addressing whether specific members of the basic leucine zipper family (BZIP) play critical roles in defining the site of alpha gene expression. We will also examine other transcription factors and determine how their targeted reduction affects expression of alpha and other genes expressed in trophoblasts and gonadotropes.
In Aim 3, we focus on the LHbeta gene and use both transfection and transgenic approaches to define elements that are both necessary and sufficient for gonadotrope-specific expression in vivo. In addition, we will use transgenic mice to explore the importance of specific transcription factors to gonadotrope health in vivo. Pursuit of these aims will involve a variety of approaches including: cloning of new transcriptional factors; transient transfection paradigms with dominant negative constructs that prevent occupancy of a target cis-acting elements; construction of stable """"""""knockdown"""""""" cell lines that lack specific trans-acting factors; and ultimate validation of physiological significance with transgenic mice that harbor dominant-negative genes targeted to gonadotropes. In essence, we intend to establish a hierarchy of genes that define functional properties of trophoblasts and gonadotropes. Such knowledge will spawn development of new therapeutic tools that include both agents and targets.
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